Field of the Invention
The present invention relates to a method of obtaining natural blue anthocyanin-containing colorant compositions by selectively isolating fractions of anthocyanin molecules from anthocyanin-containing vegetable and fruit juices and extracts.
Description of the Related Art
There is increasing interest in the food industry to replace synthetic materials for coloring foods with natural colorants.
One challenge in replacing synthetic colorants with natural colorants has been identifying natural colorants that provide color characteristics similar to those provided by synthetic colorants.
Natural colorants that provide the same color characteristics as the synthetic blue colorant, FD&C Blue No. 1, have not been found, to this time. The lack of appropriate natural cyan blue hue colorants has also made it challenging to obtain desired natural green hue colorants from the blending of natural blue and yellow colorants. Spirulina Blue, a blue-green algae-derived material, is used as a natural blue colorant, but does not provide the same color characteristics as FD&C Blue No. 1.
Anthocyanins are water-soluble compounds found in the cell vacuoles of fruits, vegetables, and flower petals, and sometimes, roots, leaves, stems, and bracts of plants. At least in part due to their wide availability, anthocyanin-containing vegetable and fruit juices and extracts have been used as natural, edible colorants and to produce colorant compositions, in particular, natural red, purple, and blue hue colorant compositions.
An anthocyanin comprises an anthocyanidin (the aglycone) esterified to one or more sugar molecules (the glycone(s)) to form a glycoside. Sugar molecules may be attached at the C-3, C-5, C-7, C-3′, C-4′, and/or C-5′ positions. Examples of sugar molecules found in anthocyanin structures are arabinose, galactose, glucose, rhamnose, rutinose, sambubiose, sophorose, and xylose.
Anthocyanins may also be acylated, i.e., they may have one or more molecules esterified to the sugar molecules, typically at the 6-position of a monosaccharide, but also potentially at the 2-, 3-, or 4-positions. The most common acyl units include those derived from coumaric, ferulic, caffeic, sinapic, gallic, malonic, acetic, malic, succinic, vanillic, and oxalic acids.
The structure of an anthocyanidin is shown below in the flavylium cation form, which is the primary form under acidic conditions. The anthocyanidin may be substituted with hydrogen, hydroxyl, and/or methoxyl groups at various positions:
wherein R3 is H or OH,                R5 is H, OH, or OCH3,        R6 is H or OH,        R7 is OH or OCH3,        R3′ is H, OH, or OCH3,        R4′ is OH or OCH3, and        R5′ is H, OH, or OCH3.        
The most common anthocyanidins in nature are shown by the following structures:

Therefore, the class of compounds known as anthocyanins encompasses an enormous number of structurally diverse compounds based on differences in primary structure, glycosylation and acylation patterns.
Known plant sources of anthocyanins include: (1) vegetables such as red cabbage, purple sweet potato, blue potato, red potato, red radish, black carrot, purple carrot, purple corn, red corn, red onion, purple broccoli, red broccoli, purple cauliflower, rhubarb, black bean, red leaf lettuce, black rice and eggplant; and (2) fruits such as strawberry, raspberry, cranberry, lingonberry, red grape, apple, black currant, red currant, cherry, blueberry, elderberry, bilberry, crowberry, blackberry, chokeberry, gooseberry, açaí, nectarine, peach, plum, blood orange and blue tomato. Each anthocyanin source contains different amounts of multiple, distinct anthocyanin species, with 15 to 30 structurally distinct anthocyanin molecules being common for a given plant source.
The color characteristics of anthocyanin-containing vegetable and fruit juices and extracts change as a result of changing pH. Anthocyanin-containing juices and extracts generally exhibit red hues at low pH, and the hue shifts to purple as the pH is increased. Only a few juices and extracts exhibit a blue hue as pH is increased further.
The change in color of anthocyanin-containing juices and extracts resulting from changes in pH is related to the numerous secondary structures of anthocyanins that may exist in equilibrium with the primary flavylium cation structure in aqueous solution. When pH is changed, the relative quantities of the different equilibrium structures will change. At a given pH, one or more structural forms may predominate, while others are present in low quantities or not present. For example, at very low pH, the flavylium cation form predominates. As pH is increased, molecules in the flavylium cation form may be deprotonated and converted to the carbinol pseudobase form, which may be further converted through loss of a water molecule and a proton to the neutral and ionized quinonoidal base forms, respectively, and further, to the chalcone form. These transformations reduce the quantity of molecules in the flavylium cation form and increase the quantities in the other equilibrium forms to different extents. Therefore, the different equilibrium structures exist in different relative quantities at higher pH compared to low pH. Each structural form of anthocyanin may absorb light differently, resulting in a different perceived color, including no color. Therefore, as the pH of the solution is changed, changes in the relative quantities of the different structural forms may result in changes in the color of the solution.
Each distinct anthocyanin molecule is characterized by its own set of equilibrium molecular structures and equilibrium constants for the reactions that transform one structure into another. For example, the reaction transforming one anthocyanin equilibrium structure into another may have a particular acid dissociation constant, Ka, associated with it. The reaction may also be discussed in terms of the logarithmic constant, pKa, which is defined as—log10 Ka.
The flavylium cation and quinonoidal base structures have conjugated bonds connecting all three rings of the anthocyanin molecules. The extensive delocalized pi bonds allow the flavylium cation and quinonoidal base to absorb visible light, resulting in the perceived red hue of the flavylium cation at low pH and the purple or blue hue of the ionized quinonoidal base at a higher pH. In contrast, the carbinol pseudobase and chalcone structures do not have delocalized pi bonds connecting all three rings and are colorless or slightly yellow.
The substitution pattern of anthocyanins also affects color. For example, it is generally observed that the hue shifts from pink to purple when hydrogen atoms are replaced with hydroxyl groups. Similarly, the number of glycosyl (sugar) units and the number and type of acyl units are observed to affect color. However, these phenomena are not well understood or predictable.
Additionally, intermolecular and intramolecular interactions also affect anthocyanin color. The same anthocyanin may produce different hues depending on the other molecules present. For example, it is believed that acyl groups on the anthocyanin sugars can fold in and protect the flavylium cation C-2 position from nucleophilic attack. Therefore, this intramolecular interaction prevents formation of the colorless carbinol pseudobase structure. Similarly, it is believed that anthocyanin molecules self-associate, which is evidenced by the fact that a two-fold increase in anthocyanin concentration can cause a 300-fold increase in chroma, and can change the hue and value as well. It is hypothesized that this self-association is similar to intramolecular stacking, and prevents nucleophilic attack and formation of the carbinol pseudobase structure.
Although it is known that factors such as pH, anthocyanin chemical structure, substituent patterns, inter- and intra-molecular interactions all impact the color observed in anthocyanin-containing vegetable and fruit juices and extracts, it is not well understood how these factors interact to alter color; i.e., the specific cause and effect are not predictable.
For example, individual anthocyanin molecules have been separated by HPLC, but the separation has always occurred at low pH, and the color characteristics of individual anthocyanins were analyzed at low pH. Similarly, the effect of pH on the color characteristics of anthocyanin-containing vegetable and fruit juices and extracts has been studied, but these studies have analyzed the complex mixtures of anthocyanins naturally occurring in the juices and extracts. How changing pH affects the color characteristics of individual anthocyanin molecules or fractions of anthocyanins separated from natural sources, however, is not well understood or predictable. The prior art discloses that the number and types of substituents, e.g., the sugar and acyl groups, impact color; however, it does not disclose and it is not known how these substituents affect color as pH changes. Finally, although the prior art hypothesizes that various inter- and intra-molecular interactions affect color, it does not disclose how changing pH affects these inter- and intra-molecular interactions and, ultimately, the observed color of the anthocyanins.
WO 2009/100165 A2 discloses a method of separating anthocyanins from other phenolic molecules in the juice of anthocyanin-containing fruits and vegetables. WO 2009/100165 A2 does not disclose selectively separating fractions of anthocyanin molecules based on differences in charge and polarity of the molecules to produce fractions with a desired color that is different than the anthocyanin-containing juice.
The separation of individual anthocyanins at analytical scale is described in J. Chromatography A., 1148 (2007), 38-45. The separation is conducted at low pH, i.e., pH of less than 2, using HPLC in order to assist in identifying individual anthocyanins. This method separates anthocyanin molecules for detection rather than producing fractions with mixtures of anthocyanins.
WO 2004/012526 discloses a blue colorant solution of red cabbage anthocyanins at a pH of 7.9 that is used in a sugar-syrup for coating confectionery cores. The red cabbage anthocyanins were not separated into fractions.
There is no example in the prior art of isolating fractions of anthocyanin molecules from anthocyanin-containing vegetable and fruit juices and extracts at a select pH based on differences in charge and polarity of the anthocyanin molecules. In addition, methods for obtaining anthocyanin fractions that provide different color characteristics than those provided by the source juices and extracts have not been disclosed. In particular, the prior art has not described a method for obtaining a natural blue anthocyanin-containing colorant composition providing color characteristics similar to those provided by the synthetic blue colorant, FD&C Blue No. 1.
It is desirable to have a broad palette of natural colorants available for coloring foods. There is a long-felt need for natural blue colorants that provide color characteristics similar to those provided by synthetic FD&C Blue No. 1. Therefore, a method of obtaining such natural blue colorants from anthocyanin-containing vegetable and fruit juices and extracts is desired.